Testing methods to diagnose TRK fusion cancer - a plain language summary and patient perspective.

WHAT IS THIS SUMMARY ABOUT?: This is a plain language summary of a 2022 study published in Cancer Genetics. This study describes the different tests used to detect a rare type of cancer called TRK (tropomyosin receptor kinase) fusion cancer in people taking part in three clinical trials testing the medication larotrectinib. Larotrectinib targets TRK fusion proteins, abnormal proteins that result from abnormal NTRK (neurotrophic tyrosine receptor kinase) gene fusions. People who were shown to have solid tumors containing TRK fusion proteins were able to participate in clinical trials that evaluated larotrectinib. WHAT WERE THE RESULTS?: Different testing methods were used to identify participants with TRK fusion cancer. Which test was used depended on different factors such as how commonly NTRK gene fusions are found in a specific cancer type, and the cost and accessibility of the test. Participants with different types of cancer were included in the study, which allowed researchers to identify which TRK fusion proteins were found across various types of tumor. WHAT DO THE RESULTS MEAN?: The results of this study provide a guidance for healthcare professionals on the methods used for testing to identify patients that have TRK fusion cancer. By characterizing the types of testing done across cancer types, patients and their caregivers can gain an understanding of the importance of testing. This plain language summary also includes insights and perspectives from a person affected by TRK fusion cancer, and from patient advocates. Clinical Trial Registration: NCT02122913 (ClinicalTrials.gov) Clinical Trial Registration: NCT02637687 (ClinicalTrials.gov) Clinical Trial Registration: NCT02576431 (ClinicalTrials.gov).

Progressing super-enhancer landscape during mammary differentiation controls tissue-specific gene regulation.

The mammary luminal lineage relies on the common cytokine-sensing transcription factor STAT5 to establish super-enhancers during pregnancy and initiate a genetic program that activates milk production. As pups grow, the greatly increasing demand for milk requires progressive differentiation of mammary cells with advancing lactation. Here we investigate how persistent hormonal exposure during lactation shapes an evolving enhancer landscape and impacts the biology of mammary cells. Employing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggesting that their activities evolve with advancing differentiation. Using mouse genetics, we demonstrate that the functions of individual enhancers within the Wap super-enhancer evolve as lactation progresses. Most profoundly, a seed enhancer, which is mandatory for the activation of the Wap super-enhancer during pregnancy, is not required during lactation, suggesting compensatory flexibility. Combinatorial deletions of structurally equivalent constituent enhancers demonstrated differentiation-specific compensatory activities during lactation. We also demonstrate that the Wap super-enhancer, which is built on STAT5 and other common transcription factors, retains its exquisite mammary specificity when placed into globally permissive chromatin, suggesting a limited role of chromatin in controlling cell specificity. Our studies unveil a previously unrecognized progressive enhancer landscape where structurally equivalent components serve unique and differentiation-specific functions.

Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice.

The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers. We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the Csn1s1 mammary enhancer from neighboring genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal and silent Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Sult1d1 promoter and several enhancers in the casein locus. Our study demonstrates that only one out of the four CTCF sites in the casein locus had a measurable in vivo activity. Studies on additional loci are needed to determine the biological role of CTCF sites associated with enhancers.

Differential cytokine sensitivities of STAT5-dependent enhancers rely on Stat5 autoregulation.

Cytokines utilize the transcription factor STAT5 to control cell-specific genes at a larger scale than universal genes, with a mechanistic explanation yet to be supplied. Genome-wide studies have identified putative STAT5-based mammary-specific and universal enhancers, an opportunity to investigate mechanisms underlying their differential response to cytokines. We have now interrogated the integrity and function of both categories of regulatory elements using biological and genetic approaches. During lactation, STAT5 occupies mammary-specific and universal cytokine-responsive elements. Following lactation, prolactin levels decline and mammary-specific STAT5-dependent enhancers are decommissioned within 24 h, while universal regulatory complexes remain intact. These differential sensitivities are linked to STAT5 concentrations and the mammary-specific Stat5 autoregulatory enhancer. In its absence, mammary-specific enhancers, but not universal elements, fail to be fully established. Upon termination of lactation STAT5 binding to a subset of mammary enhancers is substituted by STAT3. No STAT3 binding was observed at the most sensitive STAT5 enhancers suggesting that upon hormone withdrawal their chromatin becomes inaccessible. Lastly, we demonstrate that the mammary-enriched transcription factors GR, ELF5 and NFIB associate with STAT5 at sites lacking bona fide binding motifs. This study provides, for the first time, molecular insight into the differential sensitivities of mammary-specific and universal cytokine-sensing enhancers.

Cell-specific and shared regulatory elements control a multigene locus active in mammary and salivary glands.

Regulation of high-density loci harboring genes with different cell-specificities remains a puzzle. Here we investigate a locus that evolved through gene duplication and contains eight genes and 20 candidate regulatory elements, including one super-enhancer. Casein genes (Csn1s1, Csn2, Csn1s2a, Csn1s2b, Csn3) are expressed in mammary glands, induced 10,000-fold during pregnancy and account for 50% of mRNAs during lactation, Prr27 and Fdcsp are salivary-specific and Odam has dual specificity. We probed the function of 12 candidate regulatory elements, individually and in combination, in the mouse genome. The super-enhancer is essential for the expression of Csn3, Csn1s2b, Odam and Fdcsp but largely dispensable for Csn1s1, Csn2 and Csn1s2a. Csn3 activation also requires its own local enhancer. Synergism between local enhancers and cytokine-responsive promoter elements facilitates activation of Csn2 during pregnancy. Our work identifies the regulatory complexity of a multigene locus with an ancestral super-enhancer active in mammary and salivary tissue and local enhancers and promoter elements unique to mammary tissue.

Cell-specific and shared enhancers control a high-density multi-gene locus active in mammary and salivary glands.

Regulation of high-density loci harboring genes with different cell-specificities remains a puzzle. Here we investigate a locus that evolved through gene duplication 1 and contains eight genes and 20 candidate regulatory elements, including a super-enhancer. Five genes are expressed in mammary glands and account for 50% of all mRNAs during lactation, two are salivary-specific and one has dual specificity. We probed the function of eight candidate enhancers through experimental mouse genetics. Deletion of the super-enhancer led to a 98% reduced expression of Csn3 and Fdcsp in mammary and salivary glands, respectively, and Odam expression was abolished in both tissues. The other three casein genes were only marginally affected. Notably, super-enhancer activity requires the additional presence of a distal Csn3 -specific enhancer. Our work identifies an evolutionary playground on which regulatory duality of a multigene locus was attained through an ancestral super-enhancer active in mammary and salivary tissue and gene-specific mammary enhancers.

Cell-specific and shared enhancers control a high-density multi-gene locus active in mammary and salivary glands.

Regulation of high-density loci harboring genes with different cell-specificities remains a puzzle. Here we investigate a locus that evolved through gene duplication 1 and contains eight genes and 20 candidate regulatory elements, including a super-enhancer. Five genes are expressed in mammary glands and account for 50% of all mRNAs during lactation, two are salivary-specific and one has dual specificity. We probed the function of eight candidate enhancers through experimental mouse genetics. Deletion of the super-enhancer led to a 98% reduced expression of Csn3 and Fdcsp in mammary and salivary glands, respectively, and Odam expression was abolished in both tissues. The other three casein genes were only marginally affected. Notably, super-enhancer activity requires the additional presence of a distal Csn3 -specific enhancer. Our work identifies an evolutionary playground on which regulatory duality of a multigene locus was attained through an ancestral super-enhancer active in mammary and salivary tissue and gene-specific mammary enhancers.

Indian Hedgehog release from TNF-activated renal epithelia drives local and remote organ fibrosis.

Progressive fibrosis is a feature of aging and chronic tissue injury in multiple organs, including the kidney and heart. Glioma-associated oncogene 1 expressing (Gli1+) cells are a major source of activated fibroblasts in multiple organs, but the links between injury, inflammation, and Gli1+ cell expansion and tissue fibrosis remain incompletely understood. We demonstrated that leukocyte-derived tumor necrosis factor (TNF) promoted Gli1+ cell proliferation and cardiorenal fibrosis through induction and release of Indian Hedgehog (IHH) from renal epithelial cells. Using single-cell-resolution transcriptomic analysis, we identified an "inflammatory" proximal tubular epithelial (iPT) population contributing to TNF- and nuclear factor κB (NF-κB)-induced IHH production in vivo. TNF-induced Ubiquitin D (Ubd) expression was observed in human proximal tubular cells in vitro and during murine and human renal disease and aging. Studies using pharmacological and conditional genetic ablation of TNF-induced IHH signaling revealed that IHH activated canonical Hedgehog signaling in Gli1+ cells, which led to their activation, proliferation, and fibrosis within the injured and aging kidney and heart. These changes were inhibited in mice by Ihh deletion in Pax8-expressing cells or by pharmacological blockade of TNF, NF-κB, or Gli1 signaling. Increased amounts of circulating IHH were associated with loss of renal function and higher rates of cardiovascular disease in patients with chronic kidney disease. Thus, IHH connects leukocyte activation to Gli1+ cell expansion and represents a potential target for therapies to inhibit inflammation-induced fibrosis.

Redundant and non-redundant cytokine-activated enhancers control Csn1s2b expression in the lactating mouse mammary gland.

Enhancers are transcription factor platforms that synergize with promoters to control gene expression. Here, we investigate enhancers that activate gene expression several hundred-fold exclusively in the lactating mouse mammary gland. Using ChIP-seq for activating histone marks and transcription factors, we identify two candidate enhancers and one super-enhancer in the Csn1s2b locus. Through experimental mouse genetics, we dissect the lactation-specific distal enhancer bound by the mammary-enriched transcription factors STAT5 and NFIB and the glucocorticoid receptor. While deletions of canonical binding motifs for NFIB and STAT5, individually or combined, have a limited biological impact, a non-canonical STAT5 site is essential for enhancer activity during lactation. In contrast, the intronic enhancer contributes to gene expression only in late pregnancy and early lactation, possibly by interacting with the distal enhancer. A downstream super-enhancer, which physically interacts with the distal enhancer, is required for the functional establishment of the Csn1s2b promoter and gene activation. Lastly, NFIB binding in the promoter region fine-tunes Csn1s2b expression. Our study provides comprehensive insight into the anatomy and biology of regulatory elements that employ the JAK/STAT signaling pathway and preferentially activate gene expression during lactation.

Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos.

Deaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 and ABE. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls. Therefore, an optimization of cytosine base editors is required to improve its fidelity. While the remarkable fidelity of ABE has implications for a wide range of applications, the occurrence of rare aberrant C-to-T conversions at specific target sites needs to be addressed.

Enhancer and super-enhancer dynamics in repair after ischemic acute kidney injury.

The endogenous repair process can result in recovery after acute kidney injury (AKI) with adaptive proliferation of tubular epithelial cells, but repair can also lead to fibrosis and progressive kidney disease. There is currently limited knowledge about transcriptional regulators regulating these repair programs. Herein we establish the enhancer and super-enhancer landscape after AKI by ChIP-seq in uninjured and repairing kidneys on day two after ischemia reperfusion injury (IRI). We identify key transcription factors including HNF4A, GR, STAT3 and STAT5, which show specific binding at enhancer and super-enhancer sites, revealing enhancer dynamics and transcriptional changes during kidney repair. Loss of bromodomain-containing protein 4 function before IRI leads to impaired recovery after AKI and increased mortality. Our comprehensive analysis of epigenetic changes after kidney injury in vivo has the potential to identify targets for therapeutic intervention. Importantly, our data also call attention to potential caveats involved in use of BET inhibitors in patients at risk for AKI.

Simultaneous targeting of linked loci in mouse embryos using base editing.

A particular challenge in genome engineering has been the simultaneous introduction of mutations into linked (located on the same chromosome) loci. Although CRISPR/Cas9 has been widely used to mutate individual sites, its application in simultaneously targeting of linked loci is limited as multiple nearby double-stranded DNA breaks created by Cas9 routinely result in the deletion of sequences between the cleavage sites. Base editing is a newer form of genome editing that directly converts C∙G-to-T∙A, or A∙T-to-G∙C, base pairs without introducing double-stranded breaks, thus opening the possibility to generate linked mutations without disrupting the entire locus. Through the co-injection of two base editors and two sgRNAs into mouse zygotes, we introduced C∙G-to-T∙A transitions into two cytokine-sensing transcription factor binding sites separated by 9 kb. We determined that one enhancer activates the two flanking genes in mammary tissue during pregnancy and lactation. The ability to introduce linked mutations simultaneously in one step into the mammalian germline has implications for a wide range of applications, including the functional analysis of linked cis-elements creating disease models and correcting pathogenic mutations.

Targeting fidelity of adenine and cytosine base editors in mouse embryos.

Base editing directly converts a target base pair into a different base pair in the genome of living cells without introducing double-stranded DNA breaks. While cytosine base editors (CBE) and adenine base editors (ABE) are used to install and correct point mutations in a wide range of organisms, the extent and distribution of off-target edits in mammalian embryos have not been studied in detail. We analyze on-target and proximal off-target editing at 13 loci by a variety of CBEs and ABE in more than 430 alleles generated from mouse zygotic injections using newly generated and published sequencing data. ABE predominantly generates anticipated A•T-to-G•C edits. Among CBEs, SaBE3 and BE4, result in the highest frequencies of anticipated C•G-to-T•A products relative to editing byproducts. Together, these findings highlight the remarkable fidelity of ABE in mouse embryos and identify preferred CBE variants when fidelity in vivo is critical.

Lineage-Specific and Non-specific Cytokine-Sensing Genes Respond Differentially to the Master Regulator STAT5.

STAT5, a member of the family of signal transducers and activators of transcription, senses cytokines and controls the biology of cell lineages, including mammary, liver, and T cells. Here, we show that STAT5 activates lineage-specific and widely expressed genes through different mechanisms. STAT5 preferentially binds to promoter sequences of cytokine-responsive genes expressed across cell types and to putative enhancers of lineage-specific genes. While chromatin accessibility of STAT5-based enhancers was dependent on cytokine exposure, STAT5-responsive promoters of widely expressed target genes were generally constitutively accessible. While the contribution of STAT5 to enhancers is well established, its role on promoters is poorly understood. To address this, we focused on Socs2, a widely expressed cytokine-sensing gene. Upon deletion of the STAT5 response elements from the Socs2 promoter in mice, cytokine induction was abrogated, while basal activity remained intact. Our data suggest that promoter-bound STAT5 modulates cytokine responses and enhancer-bound STAT5 is mandatory for gene activation.

Hierarchy within the mammary STAT5-driven Wap super-enhancer.

Super-enhancers comprise dense transcription factor platforms highly enriched for active chromatin marks. A paucity of functional data led us to investigate the role of super-enhancers in the mammary gland, an organ characterized by exceptional gene regulatory dynamics during pregnancy. ChIP-seq analysis for the master regulator STAT5A, the glucocorticoid receptor, H3K27ac and MED1 identified 440 mammary-specific super-enhancers, half of which were associated with genes activated during pregnancy. We interrogated the Wap super-enhancer, generating mice carrying mutations in STAT5-binding sites within its constituent enhancers. Individually, the most distal site displayed the greatest enhancer activity. However, combinatorial mutation analysis showed that the 1,000-fold induction in gene expression during pregnancy relied on all enhancers. Disabling the binding sites of STAT5, NFIB and ELF5 in the proximal enhancer incapacitated the entire super-enhancer. Altogether, these data suggest a temporal and functional enhancer hierarchy. The identification of mammary-specific super-enhancers and the mechanistic exploration of the Wap locus provide insights into the regulation of cell-type-specific expression of hormone-sensing genes.

Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster.

The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.

dbVar structural variant cluster set for data analysis and variant comparison.

dbVar houses over 3 million submitted structural variants (SSV) from 120 human studies including copy number variations (CNV), insertions, deletions, inversions, translocations, and complex chromosomal rearrangements. Users can submit multiple SSVs to dbVAR  that are presumably identical, but were ascertained by different platforms and samples,  to calculate whether the variant is rare or common in the population and allow for cross validation. However, because SSV genomic location reporting can vary - including fuzzy locations where the start and/or end points are not precisely known - analysis, comparison, annotation, and reporting of SSVs across studies can be difficult. This project was initiated by the Structural Variant Comparison Group for the purpose of generating a non-redundant set of genomic regions defined by counts of concordance for all human SSVs placed on RefSeq assembly GRCh38 (RefSeq accession GCF_000001405.26). We intend that the availability of these regions, called structural variant clusters (SVCs), will facilitate the analysis, annotation, and exchange of SV data and allow for simplified display in genomic sequence viewers for improved variant interpretation. Sets of SVCs were generated by variant type for each of the 120 studies as well as for a combined set across all studies. Starting from 3.64 million SSVs, 2.5 million and 3.4 million non-redundant SVCs with count >=1 were generated by variant type for each study and across all studies, respectively. In addition, we have developed utilities for annotating, searching, and filtering SVC data in GVF format for computing summary statistics, exporting data for genomic viewers, and annotating the SVC using external data sources.

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications.

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.